Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. Our long-term objectives are to understand the molecular virology of HCV and translate this knowledge into new antiviral strategies. Like other positive-strand RNA viruses, HCV is believed to replicate in association with cytoplasmic membranes, although the mechanistic details remain unknown. NS5A, one of the virus'non-structural proteins appears to play a key role in this process. How NS5A is localized to its restricted subset of host cell cytoplasmic membranes has been provocative considering it lacks features commonly responsible for the membrane association of proteins. An amphipathic helix (AH) has recently been identified in the N-terminal domain of NS5A. This structural motif is necessary and sufficient for membrane localization. An amphipathic helix is conserved across HCV isolates. Genetically disrupting the amphipathic helix-mediated localization of NS5A impairs replication of HCV RNA. These results have exciting implications with respect to the role of NS5A in the HCV life cycle, HCV's interaction with its host cell, and the design of novel anti-HCV therapies. We hypothesize: 1) the minimal elements required for membrane association or RNA replication can be further narrowed to a subset of amino acids within the AH;2) the association is mediated in part by a protein partner in the targeted membranes;3) mislocalization of NS5A affects the assembly of other components of the RNA replication complex;4) an HCV genome encoding a mutated NS5A AH can be complemented in trans;5) the membrane association of NS5A can be pharmacologically disrupted. We will test these hypotheses by using mutagenesis, immunomicroscopy, and in vitro membrane association assays to identify the critical viral and host features required for NS5A membrane association. These methods will also be used to determine how other components of the replicase complex are linked to NS5A's membrane association. Molecular genetics with HCV replicons will be used to define NS5A's cis and trans functions in RNA replication. We will use phage display to identify high affinity random peptide ligands of the AH. Finally, we will explore how our results can be translated into novel anti-HCV strategies.